Far away from home? Ancient DNA shows the presence of bicolored shrew (Crocidura leucodon) in Bronze Age Denmark

Abstract An excavation of an Early Iron Age village near Aalborg in Denmark uncovered the jaws and skull fragments from a small mammal that were morphologically identified to the genus Crocidura (white‐toothed shrews). Three Crocidura species are known from prehistoric continental Europe but none of them are distributed in Scandinavia, which is why this surprising finding warranted further analyses. The bone was radiocarbon‐dated to 2840–2750 calibrated years before present (cal. BP), corresponding to the Late Bronze Age and hence earlier than the Iron Age archeological context in which it was found. Using highly optimized ancient DNA protocols, we extracted DNA from one tooth and shotgun‐sequenced the sample to reconstruct a near‐complete mitochondrial reference genome (17,317 bp, 32.6× coverage). Phylogenetic analyses determined this specimen as a bicolored shrew (Crocidura leucodon) but with a phylogenetic position basal to the clade of known sequences from this species. The confirmation of Crocidura presence in Denmark by the Late Bronze Age sheds new light on the prehistoric natural history of Scandinavia. We discuss the implications of this finding from both zoo‐archeological and ecological perspectives. Furthermore, the mitochondrial genome reconstructed in this study offers a valuable resource for future research exploring the genetic makeup and evolutionary history of Eurasian shrew populations.

prehistoric coastline indicate that the houses were placed close to the ancient shore, where it is now located more than 5 km from the Limfjord (Kristiansen et al., 2021).
Zoological remains were also recovered from the site, including c. 600 bones from micromammals (Table S1).One particularly unexpected zoological discovery was the identification of two jawbones and skull fragments from the white-toothed shrew genus (Crocidura) of which there are no confirmed recordings from Denmark.The exact species could not be determined confidently from morphological analyses, but it was evident that this was a highly unusual specimen.In addition to filling important knowledge gaps on prehistoric Scandinavian biodiversity, this discovery may hold implications for understanding biodiversity responses to a changing environment.It is widely recognized that small mammals serve as crucial indicator species for monitoring environmental changes, particularly climatic conditions, over time (Hope et al., 2017;Rowe & Terry, 2014).
Conversely, analyses of animal bones that are found severely "out of place" can be key to understanding why some bone assemblages do not accurately reflect the local fauna composition.Such mechanisms could, for example, involve rare long-range dispersals (Moll et al., 2021;Mukhacheva & Tolkachev, 2022), biased fossil deposition (Allentoft et al., 2010;Kattel et al., 2007), or humanor animal-mediated movement of certain species or their remains (Brace et al., 2016;Bullock et al., 2018).By recognizing the importance and uniqueness of the identified Crocidura specimens, we conducted a series of analyses to identify their age and taxonomy.
Shrews are members of the family Soricidae and constitute a group of small mammals that mainly feed on insects and other small invertebrates.Shrews show a high sensitivity to harsh environmental conditions, particularly during the winter season.This is due to their rapid metabolism, small body size, and limited capacity to store energy in the form of fat reserves (Dokulilová et al., 2023).
The Soricidae family encompasses three subfamilies including Crocidurinae (white-toothed shrews), Soricinae (red-toothed shrews) and Myosoricinae.Myosoricinae is confined to Africa and represents <1% of the all known shrew species but the other two subfamilies have a diverse assemblage of species distributed across the globe from forests to grasslands and wetlands (Neves et al., 2019;Von Merten & Siemers, 2012).
Europe is home to a large diversity of shrew species from the genera Neomys, Sorex (red-toothed shrews) and Crocidura (white-toothed shrews).The genus Neomys encompasses the water shrews, which are known for their semi-aquatic lifestyle and adaptations to life near freshwater habitats.Key morphological characteristics of these shrews include larger hind feet fringed with stiff hairs, which extend to their toes, fingers and tail.These fringes serve to increase surface area, improve propellant power, and enhance stability during swimming (Krystufek et al., 2000).The genus Sorex is typically terrestrial (Churchfield & Rychlik, 2006), and is broadly distributed in Eurasia and North America (Mackiewicz et al., 2017).Despite similar diet, there are significant mandible muscular and skeletal morphology variations within this genus (Young et al., 2010).The Crocidura genus not only differs from its red-toothed family members in morphology and tooth formula but also lacks pigmentation in its teeth, hence earning its common name, i.e., the white-toothed shrews.They are the most species-rich genus among all mammals, currently comprising more than 200 recognized species, many of which are difficult to differentiate solely based on morphological characters (Li et al., 2023).Therefore, additional analyses, such as biochemical, cytogenetic and molecular tests are often necessary to obtain a secure species identification (Rofes & Cuenca-Bescos, 2011).
In Europe, the three most widespread species of Crocidura include the lesser white-toothed shrew (Crocidura suaveolens), the bicolored white-toothed shrew (Crocidura leucodon), and the greater whitetoothed shrew (Crocidura russula) (Neves et al., 2021).However, despite their widespread distribution in Europe, in Denmark, shrews from the genus Crocidura have been recorded only once in the remains of a barn owl pellet (Laursen, 2024), and never reported from prehistoric material.The species of shrew found in Denmark (i) determine its chronological age, (ii) obtain a secure species identification, and (iii) establish its phylogenetic position in the context of other shrews.

| Archeological context and sample description
The skull and jawbone fragments of the Crocidura species (specimen's museum number: ÅHM 6023 ×5543) was found within a small building, A14522, in a waste layer of bones and sherds upon a lime floor (Figure 1).The floor measures 4 × 4 m and was surrounded by stones to the north and a large intact pavement to the west.The pavement west of A14522 represents the southern entrance area of longhouse A15624, with a stable in the eastern part, a living area in the western part, and two opposing doors in the middle of the longhouse.The farm thereby consists of a 16-m-long longhouse, a well-preserved stone-paved entrance area and a smaller outbuilding placed to the south of the stable.The bone fragments emerged during wet sieving of the waste layer using net sizes of 5 mm and 2 mm (applied to 90% and 10% of the samples, respectively).Initial taxonomic identification of the remains was based on dental formula, bone and tooth morphology (Hutterer, 2005;Niethammer & Krapp, 1990;Richter, 1964;Vogel et al., 1989).

| Radiocarbon age determination
The radiocarbon age determination was conducted at the Aarhus AMS Centre.The collagen extraction procedure followed a modified version of the widely used Longin Method (Brown et al., 1988;Longin, 1971).A more detailed description is available in Kveiborg and Olsen (2023).Due to the very small sample size (20.59mg) of the jawbone we extracted collagen without using NaOH and Ultrafiltration (UF), since especially the latter reduces the collagen yield considerably (Jørkov et al., 2006).After collagen extraction, we ended up with 0.46 mg of freeze-dried collagen, which we transferred to quartz tubes with 200 mg pre-cleaned CuO and subsequently evacuated, sealed and combusted to CO 2 at 900°C.Graphitization followed the H 2 reduction method using an iron catalyst and MgClO 4 to trap excess water (Santos et al., 2007;Vogel et al., 1984).A very small amount of graphitized Carbon (0.14 mg) was mounted and the measurement took place at the Aarhus AMS Centre (AARAMS) using a HVE 1 MV tandetron accelerator AMS system (Olsen et al., 2017).We report the 14 C age in conventional radiocarbon years BP (before present = 1950) in accordance with international convention (Stuiver & Polach, 1977).
To ensure a sufficient amount of carbon for the radiocarbon age determination all the collagen was used, which left no collagen for isotope analysis.The species is well known to be 100% terrestrial, so reservoir correction of the date should not be necessary.For calibration of the 14 C age we use the IntCal 20 (Reimer et al., 2020) and the Oxcal v4.4.4 calibration software (Ramsey, 2009).The probability method calculates the calibrated age ranges corresponding to 68.2% probability (1 σ) and 95.4% probability (2 σ) indicating the probability ranges of the true date.

| Ancient DNA extraction, library preparation, and sequencing
All the pre-PCR work was performed in sterile cleanlab facilities at the Lundbeck Foundation GeoGenetics Centre (Globe Institute, University of Copenhagen).One tooth, a lower (1st) incisor, was removed from the jawbone and crushed.A volume of 2 mL digestion buffer (pr.mL: 929 μL of 0.5 M EDTA, 10 μL of TE buffer, 10 μL of Proteinase K, 50 μL of 10% N-laurylsarcosine, and 1 μL of phenol red) was added to the sample and incubated for 24 h at 42°C.The DNA was then purified using the silica-in-solution method similar to Rohland and Hofreiter (2007) but using the optimized binding buffer from Allentoft et al. (2015).The final elution was performed with 50 μL TEB buffer.Double-stranded blunt-end libraries were constructed from the extracted DNA using NEBNext DNA Prep Master Mix Set E6070 (New England Biolabs Inc.) with protocol modifications (Allentoft et al., 2015;Orlando et al., 2013), and amplified with double-indexed Illumina-specific adapters prepared as Meyer and Kircher (2010).The DNA concentration of the library was quantified on an Agilent 2200 Tapestation, and pooled equimolarly with other libraries (from different projects) before sequenced on an Illumina NovaSeq6000 platform (150 PE) at the Danish National High-throughput DNA Sequencing Centre.Negative controls were included at both the extraction and library steps, yielding undetectable DNA concentrations.

| Bioinformatics
The raw sequencing data were base-called using the Illumina software CASAVA 1.8.2 and sequences were de-multiplexed with a requirement of full match of the nucleotide index sequences.The raw sequences were quality checked with FastQC (Andrews, 2010).
Adapter sequences and reads with phred quality score below 30 were removed, overlapped paired-end reads merged, and any merged reads less than 30 bp were discarded using AdapterRemoval2 (Schubert et al., 2016).To preliminarily determine if the specimen was more related to Sorex, Neomys or the Crocidura genus trimmed sequence reads were mapped against multiple mitochondrial reference genomes using BWA aln (Li & Durbin, 2009), with default parameters.The complete list of mitochondrial genomes used for mapping is available in Table 1.Reads with mapping quality lower than 25 and potential PCR duplicates were removed from the alignment by SAMtools.(Li et al., 2009) The assembly of mitogenome sequences was performed using MITObim v1.9.1 (Hahn et al., 2013) in quick mode with mismatch 2, using the following complete mitogenome references as bait: Crocidura russula, Crocidura tanakae, Crocidura sibirica, Crocidura.negrina, Crocidura shantungensis.The accession number of these mitogenomes is provided in Table 1.These references were chosen to cover a varying phylogenetic distance (within the Crocidura genus) and based on their availability on GenBank at the time.All the trimmed sequences were then re-mapped with BWA aln against each of these draft assemblies (allowing n6 and n7 mismatches) and the resulting bam-files were inspected visually in Geneious Prime 2022.1.1 (https:// www.genei ous.com).Assemblies that yielded regions with low coverage in this re-mapping exercise were excluded at this stage.Two assemblies performed well and were highly congruent in the re-mapping exercise and these were aligned (for both n6 and n7; i.e., a total of four assemblies) to construct a single consensus sequence serving as our reference assembly.As a final check for equal mitogenome-wide coverage, trimmed sequences were mapped again to this consensus assembly using BWA aln (same parameters as above).Mitogenome annotation was performed on this final assembly using MitoZ (Meng et al., 2019) and the annotations were manually checked and curated in Geneious.To assess the ancient DNA authenticity of the mapped reads, MapDamage2 (Jónsson et al., 2013) was run using the assembly as reference.

| Phylogenetics
For the phylogenetic analysis we retrieved from GenBank 23 and 21 complete or near-complete Cytb and COI mitochondrial sequences of shrew species with lengths of 1140 and 1545 bp, respectively (Table S2), including the known shrew species from Denmark (Sorex araneus, Sorex minutus, Neomys fodiens).The same regions were extracted from our own consensus sequence and aligned in Geneious Alignment (https:// www.genei ous.com), using default parameters.jModelTest v2.1.10( Darriba et al., 2012) was employed to find the best evolutionary model of nucleotide substitution in this alignment.
Two independent phylogenetic analyses were performed using these two genes.We performed a Bayesian Inference (BI) analysis in MrBayes v3.2.6 (Huelsenbeck & Ronquist, 2001) using a GTR invgamma model with nucleotide sites partitioned for 1 million generations sampling every 500 generations and a 100,000 burn-in length.

| Morphological assessment
The Crocidura findings from this site consist of a partly preserved calvarium and a left and right mandible respectively (Figure 2).The white-toothed shrews have three upper unicuspid teeth and the dental formula: 3 1 1 3 2 0 1 3 and are thus easily distinguished from other species of insectivores (Hutterer, 2005).Based on the size, the remains were deemed on morphological grounds to most likely represent C. leucodon or C. russula, which are of similar size, whereas C. suaveolens is rather smaller.Based on specific diagnostic landmarks on the mandible (processus angularis) as described by Niethammer andKrapp (1990), andRichter (1964), an initial identification to C. russula was suggested.However, this identification was contradicted by the morphology of the skull especially the shape of the upper P4 and a short rostrum, which points toward C. leucodon (Niethammer & Krapp, 1990).Other diagnostic features such as the width of the infraorbital bridge (cf.Vogel et al., 1989) and size of the upper unicuspid teeth (Niethammer & Krapp, 1990)  TA B L E 1 List of reference genomes used for mapping and building initial mitogenomes.

| Bioinformatics
We obtained 136,246,905 raw sequencing reads, of which 133,699,835 were retained after quality filtering.These were initially mapped against seven shrew mitochondrial genomes including representatives of the Crocidura genus and both the genera known from Denmark (Neomys and Sorex).The number of uniquely mapped reads (i.e., mapped to only one location on the reference mitochondrial genome) with duplicates removed is presented in Table 1.The Neomys and Sorex species had lower number of mapped reads (<250) compared with members of the Crocidura genus (>775) suggesting that our sample had higher genetic affinity with the latter.

The mitochondrial genome assemblies using five different
Crocidura sequences as reference baits resulted in draft assemblies with slightly varying sequence length (16,395 to 17,589 bp).When re-mapping our data against each of these five draft assemblies the visual inspection of the bam-files revealed multiple low coverage areas within the assemblies that were constructed with baits from C. sibirica, C. negrina, and C. shantungensis.These three assemblies were therefore not used to construct the final consensus assembly.
The consensus created from the alignment of the two assemblies resulting from using C. tanakae and C. russula as baits respectively (with 99.5% pairwise identity) had the total length of 17,317 bp.The final average coverage obtained from remapping of sequences to this final consensus was 32.6X.
The mitogenome annotation resulted in finding all 37 genes expected in animal mitogenomes, including 13 protein coding, 22 tRNA and 2 rRNA genes (Figure 4).No stop codon was seen in the coding regions of the protein-coding gene.This new mitogenome sequence and annotation were deposited in GenBank (GenBank accession number: OR951920).
The authenticity of our DNA (the mapped reads) was supported by clear sign of C-T deamination damage at the 5′ termini, observed by 30% increase in C to T transitions at that position (Table S3;

| DISCUSS ION
The unexpected identification of a bicolored shrew (C.leucodon) from the Late Bronze Age in Denmark not only highlights a remarkable zoological discovery but also raises questions about the historical distribution of this species in continental Scandinavia.
While a skull and mandible from a modern white-toothed shrew (Crocidura sp) was discovered in 2004 through the study of tens of thousands of micromammalian bones from barn owl pellets (Laursen, 2024), our ancient DNA analysis is the first to confirm the presence of C. leucodon in Denmark, and the first recorded presence of any member of the genus in this region in prehistoric times.
Today C. leucodon occurs in Europe and western Asia from France to the Caspian Sea (Shenbrot et al., 2021) (Figure 6).At its northern range, C. leucodon is broadly confined by the Kiel Canal (Nord-Ostsee-Kanal) in Germany (Borkenhagen, 1993;Shenbrot et al., 2021), and is synanthropic, preferring artificial landscapes (gardens/fields) and houses feeding on invertebrates.It is, however, capable of living in a range of habitats from moist regions characterized by dense plant cover to damp environments in the mountain and open agricultural landscapes (Shenbrot et al., 2021;Vohralík et al., 2007).(Svenning et al., 2015).Shrews, being highly sensitive to environmental conditions (Dokulilová et al., 2023), might have adjusted their ranges for example in response to climate change (Moritz & Agudo, 2013).We note that the transition between the Bronze Age and Iron Age in Northern Europe (at c. 800 BC) was marked by a shift from a relatively warm and dry to a wetter and colder climate (Barber et al., 2004;Bond et al., 1997;van Geel et al., 1996) which may have affected the C. leucodon distribution.
The phylogenetic placement of the ancient sample representing a basal split in the clade could be an effect of its age (genetic separation in time) or it could represent a distinct and now extinct local population that may have thrived in the region during the Late Bronze Age era.However, we note that higher sampling density across a wider range of the current distribution would be needed to clarify that.
While the bicolored shrew may have been more common in Bronze Age Denmark its remains are rarely found.This scarcity could be attributed to the selective preservation of certain bones over others or biased fossil deposition (Allentoft et al., 2010;Kattel et al., 2007), or the lack of fine-meshed screening of sediments, and lack of scholarly attention, as well as spatial variability in the existence of paleontological sites (Polly, 2019) which might contribute to an assemblage that does not accurately represent the true composition of the local fauna.However, this observation of a single archeological specimen of a bicolored shrew in Denmark also calls for alternative hypotheses.One such explanation could be rare long-range dispersal events, such as those reported in larger mammals (Moll et al., 2021).However, the mean dispersal progress of C. russula and C. leucodon in northern Germany was recorded about 2.5 km per year (Frank, 1984), and in Switzerland, the mean progress recorded is even smaller, at about 1 km per year (Vogel et al., 2002).
The estimated distance between the Kiel Canal in Germany (i.e., the most northern distribution of the species), and where the species remains were found in Aalborg, Denmark is about 370 km.Based on these facts, it seems highly unlikely that long range migration is an explanation.
Moreover, human-or animal-mediated movement of species or their remains introduces the possibility of intentional or unintentional transportation (Bullock et al., 2018;Poulakakis et al., 2005), potentially distorting the spatial and temporal context of the bone discovery.
While we cannot completely rule out that transportation scenario, we find it improbable that the species was eaten by a migrating bird or carnivorous mammal somewhere south of Denmark before ending up near Aalborg.We are confident that our finding could not have been from a barn owl pellet, as barn owl is a late immigrant and was not present in Denmark before 1830s (Laursen, 2006).In light of the discussion above, we favor the hypothesis that the bicolored shrew was of local origin and thus part of the local fauna.The shift toward a wetter and colder climate in Denmark after the Bronze Age (Barber et al., 2004, Bond et al., 1997, van Geel et al., 1996) may explain why we do not have any finds of this species after that period.
The finding of a shrew bone dating to the Late Bronze Age in an excavated village from the Early Iron Age raises intriguing questions

CO N FLI C T O F I NTE R E S T S TATE M E NT
There are no conflicts of interest.
today include Common shrew, also known as Eurasian shrew (Sorex araneus), Eurasian pygmy shrew (Sorex minutus), and Eurasian water shrew (Neomys fodiens) (Arter.dk,2023).Considering the significance of discovering prehistoric Crocidura remains in Scandinavia, our study employs a variety of analyses to: F I G U R E 1 (a) Map of Limfjord Area, Denmark, elevation model using LiDAR.Meadows and wetlands marked in green.The find spot is marked with a red dot.(b) The south-eastern part of Aalborg with the excavation area marked in red.The Limfjord, dividing Vendsyssel from the rest of Jutland can be seen at the top of the photo.© Region Nordjylland 2018.(c) Excavation of the small building, A14522, with a partly preserved lime floor exposed.The Crocidura bones were recovered from wet sieved soil samples within the building.© North Jutland Museums.
are of intermediate forms or difficult to assess due to fragmentation and loss of teeth (Figure 2).Based on this, the biogeographical range and history of white-toothed shrews in Northern Europe we sampled one of the mandibles for aDNA analysis and radiocarbon dating.F I G U R E 2 Skull (a + b) and right mandible (c + d) of bicolored shrew, Crocidura leucodon from Bronze Age, Denmark.(a) ventral view, (b) lateral view, (c) lingual view, and (d) lateral view.Photographs: Jonas Ogdal Jensen, Moesgaard Museum.

Figure
Figure S1).Our phylogenies estimated by MrBayes and IQ-Tree were highly congruent for both Cytb and COI genes; the three genera (i.e., Crocidura, Neomys and Sorex) and all the species were segregated as expected.Therefore, only Bayesian Inference (BI) gene trees are shown here, whereas maximum likelihood (ML) trees are included in the Figure S2.Both analyses confidently (i.e., with Bayesian posterior probability of 1.0 for both genes, ML bootstrap of 99 (Cytb) and 100 (COI)) placed the ancient shrew sample together with C. leucodon (Figure 5) but on a separate branch than the previously published C. leucodon sequences.While the pairwise identity in alignment matrix (as calculated in Geneious) among the modern C. leucodon individuals was very high, ranging from 99.4% to 99.8% in COI and 99.1% to 99.8% in cytB, the ancient shrew proved slightly differentiated from the modern ones with pairwise identities ranging from 96.9% to 97.2% (COI) and 96.5% to 96.8% (cytB).

F I G U R E 4
Circle plot visualization of the genome annotation.Inner blue ring depicts the read coverage.Numbers refer to kilobase pair positions.Green, red and yellow colors show position of protein coding, tRNA and rRNA genes, respectively.According to the Crocidura records available in IANUS(Heinrich et al., 2016), a database of published zooarchaeological data from more than 8000 European archeological sites dated from the Late Pleistocene to Early Modern Times(Heinrich et al., 2016), species of Crocidura have been reported in Belgium, Switzerland, the Czech Republic, Germany, Spain, France, Great Britain, Greece, Hungary, Italy, Moldova, the Netherlands, Portugal, and Ukraine.This is while C. leucodon was only recorded in Germany, France, Greece, Hungary, and Ukraine.Similarly, C. leucodon was neither reported in the comprehensive overview of prehistoric species published byAaris- Sørensen (2009) nor in the GBIF data base (https:// arter.dk/ landi ng-page) for Denmark.

F
Bayesian phylogenetic tree derived from Cytb (a) and COI (b) dataset.Branch numbers refer to the Bayesian posterior probability values.The trees were generated using MrBayes 3.2.6 and visualized in FigTree v1.4.4 rooted via midpoint rooting.Our ancient shrew sample is here denoted "Unknown".The discovery of an archeological specimen of C. leucodon in a region where it no longer exists today triggers a debate on potential factors contributing to such occurrences.One likely scenario is that it may have been part of a fauna that existed during periods of different climatic conditions.Past climate changes, such as fluctuations in temperature and precipitation significantly influenced the distribution and composition of ecosystems

F
I G U R E 6 Current distribution map of Crocidura leucodon, as shown in Map of Life (MOL), accessed December 2023 at https:// mol.org/ speci es/ .aboutthis temporal discrepancy.Several factors could have contributed to this.First, there might have been a continuous habitation or reuse of the site over an extended period(Attema et al., 2019), leading to a mixing of sediments and remains from different time periods.Alternatively, geological or anthropogenic processes, such as soil erosion or construction/agricultural activities(Rosentau et al., 2013), could have displaced the shrew remains from its original context.Of relevance, it must be noted that 600-900 m south of the Iron Age settlement, several pits and longhouses were excavated in an earlier campaign.Most of these features were dated to the Bronze Age according to14 C dates and finds of ceramics (Internal report, Danish national database of archeological surveys).It is likely that the fields surrounding this Bronze Age settlement include the area that later was settled in the Iron Age, and activity in the latter may have resulted in displacement of the shrew remains.In conclusion, the discovery of a C. leucodon bone from the Late Bronze Age in Denmark offers a novel perspective on prehistorical biodiversity and environmental dynamics in Scandinavia.As discussed earlier, the complexity of interpreting ancient bone assemblages emphasizes the necessity for a comprehensive approach when reconstructing the ecological history of a region.Exploring the prehistoric distribution and migration patterns of bicolored shrews in response to past environmental changes could provide valuable insights into their resilience and adaptive strategies of small mammals, particularly relevant in the context of contemporary climate change.Lastly, the mitochondrial genome reconstructed in this study serves as a valuable resource for future research, facilitating investigations into phylogenetic and population genetics in both extinct and extant shrew species.Lastly, given the importance of the first DNA-confirmed record of C. leucodon in Denmark, we suggest updating the records available on databases such as GBIF (https:// arter.dk/ landi ng-page) accordingly.AUTH O R CO NTR I B UTI O N S Mahsa Mousavi-Derazmahalleh: Formal analysis (lead); writing -original draft (equal); writing -review and editing (equal).Niels Haue: Investigation (equal); writing -review and editing (equal).Marie Kanstrup: Formal analysis (supporting); investigation (equal); writing -review and editing (equal).Jørgen T. Laursen: Formal analysis (supporting); investigation (equal); writing -review and editing (equal).Sherralee S. Lukehurst: Formal analysis (supporting); writing -review and editing (equal).Jacob Kveiborg: Conceptualization (equal); formal analysis (supporting); investigation (equal); writingreview and editing (equal).Morten E. Allentoft: Conceptualization (equal); formal analysis (supporting); investigation (equal); writingoriginal draft (equal); writing -review and editing (equal).ACK N OWLED G M ENTS This work was supported by computational resources provided by the Pawsey Supercomputing Research Centre with funding from the Australian Government and the Government of Western Australia.Sherralee S. Lukehurst acknowledges the support from the BHP-Curtin alliance within the framework of the "eDNA for Global Environment Studies (eDGES)" program.Open access publishing facilitated by Curtin University, as part of the Wiley -Curtin University agreement via the Council of Australian University Librarians.